ABSTRACT Rickettsiae are obligately intracellular bacterial pathogens transmitted by ticks, fleas, lice and mites. The clinical manifestations and morbidity of diseases caused by Rickettsia vary from mild to very severe and fatal. Rocky Mountain spotted fever is caused by Rickettsia rickettsii; it is the most prevalent rickettsiosis in the Americas and known for its malignancy. Two other rickettsioses in the Americas caused by the emerging pathogens R. philipii and R. parkeri are typically milder in most individuals. One current view of the evolution of Rickettsia species postulates that rickettsial pathogenicity does not correlate to a gain in effectors of bacterial virulence, but rather with a loss of regulatory molecules which suppress expression of virulent phenotypes. We found that a 19 kb chromosome insert in R. philipii, the nearest relative of R. rickettsi, contains genes which are associated with control and/or attenuation of virulent phenotypes in many bacteria. Some of these genes are uniquely present in R. philipii and other rickettsiae of low pathogenicity, but absent in highly virulent rickettsiae such as R. rickettsii. Therefore, our central hypothesis is that the proteins encoded by some of the insert genes may attenuate the ability of those Rickettsia which contain these elements to cause severe infections in humans. The goals of the research proposed in this application are (1) to confirm that important functions are encoded by the chromosomal insert genes in R. philipii, and (2) to determine if they indeed modulate rickettsial pathogenicity. We will test our central hypothesis by accomplishing the following Specific Aims: Aim 1. To obtain directed knockout mutants of genes encoded by the R. philipii chromosome insert. Aim 2. To assess the phenotypic differences of wild and knockout strains of R. philipii in in vitro host-cell interaction models and by comparative transcriptome analysis. With these tools we will be able to demonstrate the utility of a new genetic knockout system to inactivate selected rickettsial proteins and to conduct functional and structural studies on these proteins. These experiments will address the mechanisms of pathogenesis employed by different Rickettsia and the results may be useful in efforts to develop improved patient adjunctive therapies, and provide a novel assay pathway for screening for more effective antibiotics against rickettsioses.